Previous methods of preparing thermo-responsive cell culture supports have used two primary approaches, namely electron beam irradiation or plasma polymerization, to covalently graft poly (N-isopropylacrylamide) (pNIPAAm) chains onto tissue culture polystyrene dishes. The complicated procedures and apparatus required in these methods prevent a cost-effective adoption of this technology for specific applications. Furthermore, previous methods have generally required the use of added adhesive proteins from other individuals or other species to enhance cell attachment, and such foreign additions tend to cause immunogenic reactions of the cell sheet products after transplantation. Other methods that do not employ thermo-responsive polymers (TRPs) for detachment and harvesting of cell sheets from cell culture supports have employed proteolytic enzymes (e.g. trypsin) or mechanical agitations, which have resulted in damage to cells and their excreted extracellular matrix (ECM), thus negatively affecting their biological functions.
Nagase et al., as disclosed in J. R. Soc. Interface (2009) 6, S293-S309 and incorporated herein by reference, teach thermoresponsive micropatterned surfaces using electron beam polymerization techniques to allow the selective adhesion and growth of, for example, rat primary hepatocytes and bovine carotid endothelial cells. U.S. Pub. No. 2010/0216242 discloses a cell culture support including a polymer layer exhibiting thermoresponsiveness and a cell culture region obtained by plasma-treating a surface layer portion thereof with a reactive gas while limiting additions of cell adhesion proteins, such as collagen. However, these methods are still too expensive for wide-spread use.
Fujita et al., as disclosed in Biotechnology and Bioengineering, Vol. 103, No. 2, Jun. 1, 2009 and incorporated herein by reference, teach fabricating a cell sheet-polymer film complex involving a temperature-sensitive polymer in which cells are attached to a temperature-sensitive poly-N-isopropylacrylamide film mixed with laminin and collagen IV. As previously mentioned, added adhesive proteins are undesirable as they may deleteriously cause immunogenic reactions.
There is a need in the art for cell culture supports and methods of growing and releasing cell sheets therefrom that do not suffer from these various drawbacks. Also, the method should be simple and cost-effective to make it economically feasible for general biomedical applications.